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Acrylamide induces immediate-early gene expression in rat brain
231
Brain Research, 609 (1993) 231-236 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 18744
Acrylamide induces immediate-early gene expression in rat brain H. E n d o a, M.I. S a b r i b, J . M . S t e p h e n s c, P . H . P e k a l a c a n d S. K i t t u r a a Gerontology Research Center, Laboratory of Biological Chemistry, Baltimore, MD (USA), b Center for Research on Occupational and Environmental Toxicology, Oregon Health Sciences University, Portland, OR (USA) and c Department of Biochemistry, East Carolina University, Greenville, NC (USA) (Accepted 17 November 1992)
Key words: Acrylamide; c-los; c-jun; mRNA; Chemical injury; Neurotoxicity
Northern blot analysis was used to study the effects of acrylamide, a potent neurotoxin, on the induction of c-los and c-jun mRNA in rat brain. Male Sprague-Dawley rats (10-12 weeks old) treated with acrylamide as a single dose (100 mg/kg, i.p.) or via drinking water (0.03% w/v) for 4 weeks, were used to study acute and chronic effects on immediate-early gene expression, respectively. Acute administration of acrylamide caused a statistically significant increase in the expression of c-fos (approx. 37%) and c-jun (approx. 17%) mRNA in rat brain. By contrast, the level of c-los mRNA in chronic acrylamide treatment was not altered significantly, but the expression of c-jun mRNA was increased almost 100% as compared to control. These data show that the neurotoxin acrylamide induces immediate-early gene expression in the brain. The effects appear to be related to the route of administration, dose and duration of acrylamide treatment.
INTRODUCTION
c-fos mRNA in the ipsi- and contralateral hippocam-
The proto-oncogenes, c-fos and c-jun, and the respective proteins (Fos and Jun) are rapidly and transiently expressed in response to external stimuli in many ceils types, including neurons 32. Recent studies have shown that c-los mRNA and protein are induced in neurons and glia following brain injury 9'1°'42, hypoxia-ischemia and seizures 19 and activation of Nmethyl-D-aspartate receptor 1. c-fos has been proposed to function as a 'third messenger' molecule in the signal transduction system 32. Fos and Jun proteins are induced by neurotrophic factors, neurotransmitters, depolarizing conditions and agents that cause a voltage-gated calcium influx in PC12 cells 32. c-los is also induced in CNS neurons of intact animals by pharmacological, electrical, surgical, and physiological stimuli u. The level of c-los mRNA and corresponding protein induction is known to increase transiently in the cerebral cortex and the hippocampus after generalized seizures 31. Intrahippocampal injection of the mast cell-degranulating peptide, a bee venom component acting on the K ÷ channel, causes the expression of
pus without generating convulsions 4t. Preproenkephalin gene is known to be regulated by Fos and Jun proteins; levels of preproenkephalin mRNA were increased 1-2 h post-seizure, subsequent to the rise in Fos and Jun 47. Fos immunohistochemistry provides a cellular method to label polysynaptically activated neurons and thereby map functional pathways 38. Alterations in the structure and expression of proteins involved in signal transmission are known to be associated with experimental and human cancer 2°. The response of proto-oncogenes to xenobiotics that compromise neuronal integrity is uninvestigated. Acrylamide neurotoxicity is a suitable animal model to study the expression of these genes because responses to systemic exposure of this neurotoxin are characterized and experimentally reproducible 4,13,15,34,39,48-5°. Acute exposure leads to encephalopathy 22. Repeated intoxication with smaller doses of acylamide causes an ataxic syndrome in humans 14, neuronal loss 3'5 and distal axonal degeneration 34,48-5°. Chronically treated rodents lose microtubule-associated proteins (MAP1 and MAP2) and show dendritic changes in hippocampal
Correspondence: M.I. Sabri, Center for Research on Occupational and Environmental Toxicology (L606), Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR, USA. Fax: (1) (503) 494-4278.
232 and extrapyramidal areas ~'7. Chronic exposure also causes retrograde degeneration of long and large-diameter axons in gracile tracts and peripheral nerves, with associated sensory loss and motor weakness 39'49'50. While it is commonly suggested that axonal neuropathy results from a direct action of acrylamide on elements required for intracellular transport 2"3°'36'37'45"46,the possibility of a toxic effect on the neuronal perikaryon has been rarely investigated. This communication reports that acrylamide alters the expression of the immediate-early genes c-los and c-jun in rat brain. MATERIALS AND METHODS
Acute acrylamide treatment Sprague-Dawley rats (Charles River, Wilmington, MA) were treated with a single i.p. injection of either saline (control) or acrylamide (100 mg/kg). Rats were sacrificed by decapitation after 6 h, the brains excised, frozen on dry-ice and kept at - 80°C until used for RNA isolation. A time of 6 h was chosen for acute study since acrylamide causes optimum inhibition of fast retrograde axonal transport at this time 3°. Cerebral cortex from 5 acrylamide-treated and 3 saline-treated control rats were used for total RNA extraction.
Data were normalized to ~-actin mRNA. ¢¢-actin, an ubiquitous protein, was used as an internal control since total levels of this abundant protein were not unaltered by acrylamide. The following probes were used for these studies: (t) c-los; a l.(l-kb Pstl fragment of pfos-1 (ATCC, Rockville, MD)S; (2)c-jun; a 1.8-kb EcoRl/HindIII fragment (obtained from Dr. W.W. Lamph, The Salk Institute, San Diego)28; (3) /3-actin; 0.7-kb TaqI-Mcol cDNA fragment (Oncor, Gaithersberg, MD). RESULTS
Acute acrylamide treatment Autoradiograms of Northerns from control and acrylamide-treated samples hybridized with radiolabeled cDNA probes for c-fos, c-jun, and /3-actin are shown in (Fig. 1). Fig. 2 illustrates that the amount of c-fos mRNA (2.2 kb) was significantly increased (approx. 37%) following acrylamide treatment. The values of c-fos in acrylamide-treated (196.7 + 28.4) and control (143.2 + 9.0) samples were plotted as percent of /3-actin mRNA (Fig. 2). The values for/3-actin mRNA in acrylamide-treated (45.7 + 6.3) and control (45.8 + 0.5) samples show no significant difference in the expression of/3-actin message.
Chronic acrylamide treatment Rats were given 0.03% (w/v) acrylamide in drinking water for 4 weeks when all animals developed hind-limb weakness. Control and acrylamide-treated animals were decapitated, the brains excised, frozen on dry-ice and kept in -80°C until used for RNA extraction. Cerebral cortex from 4 acrylamide-treated and 3 saline-treated control rats were used for RNA extraction.
ACUTE ACRYLAMIDETREATMENT AC AC AC AC AC CT CT CT
RNA extraction and Northern blot analysis Brain tissues were homogenized with RNAzol B reagent (Cinna/Biotex, Friendswood, TX) (2 ml/100 mg). Homogenates were treated with 10% (v/v) chloroform, centrifuged at 12,000 × g for 15 min in a microfuge and the aqueous phase transferred to a fresh tube. An equal volume of 2-isopropanol was added, the mixture centrifuged at 12,000× g for 15 min whereupon a white-yellow pellet of RNA at the bottom of tube was obtained. The supernatant was removed, the RNA pellet washed with 75% ethanol by vortexing, and the material centrifuged for 8 rain at 7,500x g. Finally, the pellet was dissolved in diethyl pyrocarbonate-treated water. The concentration of RNA was determined spectrophotometrically by the absorbance at 260 nm. Ten micrograms of total RNA were denatured in formaldehyde and fractioned on 1% agarose gel. Size-fractionated RNA was transferred to a Nytran (Schleicher and Schuell, Keene, NH) membrane by capillary blotting and heated at 80°C. Filters were prehybridized at 46°C for 4 h in 50% (v/v) formamide containing 4 x SSC (150 mM sodium chloride, 15 mM sodium citrate), 1% SDS, 0.1% each of bovine serum albumin, polyvinylpyrrolidone, and Ficoll, 50 mM sodium phosphate, pH 7.4, and 100 tzg of yeast tRNA/ml of 0.5 mg/ml sodium pyrophosphate. Hybridization was carried out overnight in prehybridization solution containing 0.02% each of bovine serum albumin, polyvinylpyrrolidone and Ficoll, and either the c-los, c-jun, or /3-actin cDNA probe (1 × 107 cpm/ml) 4°. All cDNA probes were labeled with [32p] dATP (300 Ci/mmol) by random priming as described by Feinburg and Volgelstein12. After hybridization, the filters were washed in 0.1 x SSC, 0.1% SDS for 1 h at 65°C with constant agitation and then subjected to autoradiography using Kodak XAR-5 film in Dupont Cronex cassettes with intensifying screens for various periods at -80°C to ensure linear film response. The autoradiograms were developed and analyzed by a scanning laser densitometer. Differences in absorbance were quantified by Student's t-test. The same filters were stripped and reused for hybridization with other probes.
C-fos
c4un
9 i!ili if
~-actin
Fig. 1. Autoradiograms of RNA blot hybridized with 32p-labeled cDNA probes for c-los and c-jun and /3-actin from the cerebral cortex of acute acrylamide-treated (AC) and saline-treated control (CT) rats. Ten/zg of total RNA was loaded in each lane.
233 c-foe mRNA in acute acrylamide treatment
I
q
CHRONIC
ACRYLAMIDE
TREATMENT
AC AC AC AC CT CT CT
3oo 200 C-fos 100 ¸
=g
=_,..=
=erylamldetreated rats
controls
Fig. 2. Levels of c-los m R N A in acute acrylamide-treated and saline-treated control rats. Levels of the gene are expressed as a percent of /3-actin mRNA. Level of c-los mRNA in acrylamide treatment was significantly different from that in control ( P < 0.05). The amount of/3-actin m R N A in acrylamide-treated sample was not significantly different from controls.
Acute acrylamide treatment also significantly increased (approx. 17%) the induction of c-jun mRNA (2.7 kb and 3.2 kb) compared to control. The values of c-jun mRNA expressed as percent of/3-actin mRNA in acrylamide-treated and control samples were 74.6 + 10.9 and 63.7 + 0.1, respectively (Fig. 3).
c-jun
I~-actin
Chronic acrylamide treatment Fig. 4 shows autoradiograms of Northerns hybridized with radiolabeled c-fos, c-jun, and /3-actin cDNA probes. The level of c-fos mRNA in acrylamide-treated rats was not altered significantly compared to controls (Fig. 5). The values for c-fos mRNA in acrylamide-treated and control rats were 146.7 + 24.9 and 183.7 ___19.9, respectively (Fig. 5).
Fig. 4. Autoradiograms of RNA blot hybridized with 32P-labeled cDNA probes for c-fos and c-jun and ,8-actin from the cerebral cortex of chronic acrylamide-treated (AC) and saline-treated control (CT) rats. T e n / z g of total RNA was loaded in each lane.
By contrast, the level of c-jun mRNA in chronic acrylamide treatment (131.7 + 14.4) was significantly increased (100%) compared to control (65.9 + 11.7)
c-foe mRNA In chronic acrylamide treatment
c-jun mRNA in acute acrylamide treatment
!
3
lOO
|
80
,1, w
=
60 ,o,
o
r
, tuut=
acrylamlds, controls treated rate
Fig. 3. Levels of c-jun m R N A in acute acrylamide-treated and saline-treated control rats. Levels of the gene are expressed as a percent of fl-actin mRNA. Level of c-jun m R N A in acrylamide treatment was significantly increased compared to control ( P < 0.05). The amount of/3-actin m R N A in acrylamide-treated sample was not significantly different from controls.
-.in-
o
s©rylamldotreated rats
controls
Fig. 5. Levels of c-los m R N A in chronic acrylamide-treated and saline-treated control rats. Levels are expressed as a percent of /3-actin mRNA. Level of c-los m R N A in acrylamide-treated sample was not significantly different from that in control. The amount of fl-actin m R N A in acrylamide-treated sample was not significantly different from controls.
234
~<
200
c-jun mRNA in chronic ecrylamide treatment
U~Z
*. m Z~ Lu
-T" 100
iii ul .IL ecrylamldetreated rats
controls
Fig. 6. Levels of c-jun mRNA in chronic acr/lamide-treated and saline-treated control rats. Levels of c-jun are expressed as a percent of fl-actin mRNA. Level of c-jun mRNA in acrylamide-treated sample was significantly different from that in control (P < 0.05). The amount of fl-actin mRNA in acry]amide-treated samples was not significantly different from controls.
(Fig. 6). The values of/3-actin m R N A in chronic acrylamide-treated and control samples were 55.7 _+ 4.8 and 47.0 _+ 6.6, respectively, and are not significantly different from each other. DISCUSSION The results of this study show that systemic treatment with acrylamide induces immediate-early gene expression in rat brain. While the level of both c-los and c-jun m R N A was increased significantly following acute acrylamide treatment, only c-jun m R N A was elevated significantly following chronic acrylamide treatment. Increased mRNA could be the result of increased immediate-early gene transcription through the activation of a signal transduction system. Biochemical changes such as increased concentrations of NAD + in whole brain 25, elevated levels of 5-hydroxyindoleacetic acid in the striatum, septal area, and thalamus 35 and/3-glucuronidase activity have been reported in acrylamide-treated animals 5. Differential effects of acrylamide, given as a single injection or repeated doses, have been observed previously ~6'17. While a single injection of acrylamide produces a modest retardation of the slow axonal transport of neurofilaments, chronic acrylamide treatment causes blockade of neurofilaments, reduced axonal caliber in proximal axons, and distal axonal degeneration ~6'17. The induction of c-jun and not of c-fos after chronic treatment with acrylamide is very interesting in the light of previous studies showing selective induction of c-jun mRNA and Jun proteins in dorsal root ganglion neurons after sciatic nerve transection, crush or blockade of axonal transport with colchicine or
vinblastine 24'2~'43. These reports suggest that c-jun may play an important role in neuronal response to chemical and traumatic injury. c-los activation in neuronal cells occurs within minutes of growth factor stimulation and precedes the activation of c-myc 1~'27"33. The induction of c-fos is transient and disappears 30 min after stimulation 44. It is therefore not surprising that c-los induction is not seen following chronic acrylamide treatment. Reasons for a differential effect of acute and chronic acrylamide on the induction of immediate-early genes in rat brain is not immediately clear. However, these results should be compared with findings in ischemic animals where c-los expression was dramatically elevated 72 h post ischemic injury in the CA1 region. It was speculated that c-fos participates in the long-term alteration of cellular function in individual regions of the hippocampus following neuronal injury, and may also be involved in regeneration of nerve cell processes z6. It has been suggested that c-los plays a role in the maintenance of long-term changes by increasing synaptic activity 23. Since c-los and c-jun are thought to control physiological processes of cell growth and differentiation 21, their m R N A might be activated by acrylamide-induced pathophysiological changes. It appears therefore that acrylamide may affect neuronal signal transduction and that activation of immediate-early genes may be related to a compensatory mechanism to overcome acrylamide toxicity. In conclusion, the results of this study indicate that the expression of immediate-early genes (c-fos and c-jun) is up-regulated in the CNS following acrylamide treatment. While acute acrylamide administration induces both c-los and c-jun, chronic acrylamide treatment induces only c-jun. The results of this study suggest that IEGs may play important roles in neuronal responses to chemical injury.
Acknowledgements. We would like to acknowledge Mrs. Charlotte Adler for support with photography. Supported in part by NIH Grant NS 19611.
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235 4 Cavanagh, J.B. and Gysbers, M.F., Ultrastructural changes in axons caused by acrylamide above a nerve ligature, Neuropathol. Appl. Neurobiol., 7 (1981) 315-326. 5 Cavanagh, J.B. and Nolan, C.C., Selective loss of Purkinje cells from the rat cerebellum caused by acrylamide and the responses of fl-glucuronidase and ~-galactosidase, Neuropathologica, 58 (1982) 210-214. 6 Chauhan, N.B., Spencer, P.S. and Sabri, M.I., Effect of acrylamide on the distribution of microtubule-associated proteins (MAP1 and MAP2) in selected regions of rat brain, Mol. Chem. Neuropathol., in press. 7 Chauhan, N.B., Sabri, M.I. and Spencer, P.S., Acrylamide-induced depletion of microtubule-associated proteins (MAP1 and MAP2) in the rodent extrapyramidal system, Brain Res., in press. 8 Curran, T. and Franza Jr., B.B., Fos and Jun: the AP-1 Connection, Cell, 55 (1988) 395-397. 9 Dragunow, M., deCastro, D. and Faull, R.L.M., Induction of Fos in glia-like cells after focal brain injury but not during wallerian degeneration, Brain Res., 527 (1990) 41-54. 10 Dragunow, M., Goulding, M., FauU, R.L.M., Ralph, R., Mee, E. and Frith, R., Induction of c-fos mRNA and protein in neurons and glia after traumatic brain injury: pharmacological characterization, Exp. Neurol., 107 (1990) 236-248. 11 Dragunow, M. and Robertson, H.A., Kindling stimulation induces c-los protein(s) in granular cells of the rat dentate gyrus, Nature, 329 (1987) 441-442. 12 Feinberg, A.P. and Vogelstein, B., A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity, Addendum Anal Biochem., 137 (1984) 266-267. 13 Fullerton, P.M. and Barnes, J.M., Peripheral neuropathy in rats produced by acrylamide, Br. J. Indust. Med., 23 (1966) 210-221. 14 Fullerton, P.M., Electrophysiological and histological observations on peripheral nerves in acrylamide poisoning in man, J. Neurol. Neurosurg. Psychiatry, 32 (1969) 186-192. 15 Garland, T.O. and Patterson, M.W.H., Six cases of acrylamide poisoning, Br. Med. J., 4 (1967) 134-138. 16 Gold, B.G., Price, D.L., Griffin, J.W., Rosenfeld, J., Hoffman P.N., Sternberger, N.H. and Sternberger, L.A., Neurofilament antigens in acrylamide neuropathy, J. Neuropathol. Neurol., 47 (1988) 145-157. 17 Gold, B.G., Griffin, J.W. and Price, D.L., Slow axonal transport in acrylamide neuropathy: different abnormalities produced by single-dose and continuous administration, J. Neurosci., 5 (1985) 1755-1768. 18 Greenberg, M.E. and Ziff, E.B., Stimulation of 3T3 cells induces transcription of the c-los proto-oncogene, Nature, 311 (1984) 433-438. 19 Gunn, A.J., Dragunow, M., Faull, R.L.M. and Gluckman, P.D., Effects of hypoxia-ischemia and seizures on neuronal glial-like c-fos protein levels in the infant rat, Brain Res., 531 (1990) 105-116. 20 Herschman, H.R., Extracellular signals, transcriptional responses and cellular specificity, Trends Biochem. Sci., 14 (1989) 455-458. 21 Hisanaga, K., Sagar, S.M., Hicks, K.J., Swanson, R.A. and Sharp, F.R., c-los proto-oncogene expression in astrocytes associated with differentiation or proliferation but not depolarization, Mol. Brain. Res., 8 (1990) 69-75. 22 Igisu, H., Goto, I., Kawamura, Y., Kato, M., Izumi, K. and Kuroiwa, Y., Acrylamide encephalopathy due to well water pollution. J. Neurol. Neurosurg. Psychiatry, 38 (1975) 581-586. 23 Jeffery, K.J., Abraham, W.C., Dragunow, M. and Mason, S.E., Induction of FOS-like immunoreactivity and the maintenance of long-term potentiation in the dentate gyrus of unanesthetized rats, Mol. Brain. Res., 8 (1990) 267-274. 24 Jenkins, R. and Hunt, S.P., Long-term increase in the levels of c-jun mRNA and Jun protein-like immunoreactivity in motor and sensory neurons following axon damage, Neurosci. Lett., 129 (1991) 107-110. 25 Johnson, E.C. and Murphy, S.D., Effect of acrylamide intoxication on piridine nucleotide concentrations and functions in rat cerebral cortex, Biochem. Pharmacol., 26 (1977) 2151-2155. 26 J~argensen, M.B., Deckert, J., Wright, D.C. and Gehlert, D.R.,
Delayed corps proto-oncogene expression in the rat hippocampus induces transient global cerebral ischemia; an in situ hybridization study, Brain. Res., 484 (1989) 393-398. 27 Kruijer, W., Cooper, J.A., Hunter, T. and Verma, I.M., Plateletderived growth factor induces rapid but transient expression of the c-los gene and protein, Nature, 312 (1984) 711-716. 28 Lamph, W.W., Wamsley, P., Sassone-Corsi, P. and Verma, I.M., Induction of proto-oncogene JUN/AP-1 by serum and TPA, Nature, 334 (1988) 629-631. 29 Leah, J.D., Herdegen, T. and Bravo, R., Selective expression of Jun proteins following axotomy and axonal transport block in peripheral nerves in the rat: evidence for a role in the regeneration process, Brain Res., 566 (1991) 198-207. 30 Miller, M.S. and Spencer, P.S., Single doses of acrylamide reduce retrograde transport velocity, J. Neurochem., 43 (1984) 1401-1408. 31 Morgan, J.I., Cohen, D.R., Hempstead, J.L. and Curran, T., Mapping patterns of c-los expression in the central nervous system after seizure, Science, 237 (1987) 192-197. 32 Morgan, J.I. and Curran, T., Stimulus-transcription coupling in neurons: role of cellular immediate-early genes, Trends Neurosci., 12 (1989) 459-462. 33 Miiller, R., McCaw, P.S., Vaessin, H., Caudy, M., Jan, L.Y., Jan, Y.N., Cabrera, C.V., Buskin, J.N., Hauschka, S.D., Lassa, A.B., Weintraub, H. and Baltimore, D., Induction of c-los gene and protein by growth factors precedes activation of c-myc, Nature, 312 (1984) 716-720. 34 Prineas, J., The pathogenesis of dying-back polyneuropathies. II, An ultrastructural study of experimental acryalmide intoxication in the cat, J. Neuropathol. Exp. Neurol., 28 (1969) 598-621. 35 Rafales, LS., Lasley, S.M., Greenland, R.D. and Mandybur, T., Effects of acrylamide on locomotion and central monoamine function in the rat, PharmacoL Biochem. Behac., 19 (1983) 635644. 36 Sabri, M.I. and Spencer, P.S., Toxic distal axonopathy: biochemical studies and hypothetical mechanisms. In P.S. Spencer and H.H. Schaumburg (Eds.), Experimental and Clinical Neurotoxicology, Williams and Wilkins, Baltimore, MD, 1980, pp. 206-219. 37 Sabri, M.I. and Spencer, P.S., Acrylamide impairs fast and slow axonal transport in rat optic system, Neurochem. Res., 15 (1990) 603-608. 38 Sagar, S.M., Sharp, F.R. and Curran, T., Expression of c-los protein in brain: metabolic mapping at the cellular level, Science, 240 (1988) 1328-1331. 39 Schaumburg, H.H., Wisniewski, H.W. and Spencer, P.S., Ultrastructural studies of the dying-back process. I. Peripheral nerve terminal and axon degeneration in systemic acrylamide intoxication, J. Neuropathol. Exp. Neurol., 33 (1974) 260-284. 40 Scott, A.F., Phillips, J.A. and Migeon, B.R., DNA restriction endonuclease analysis for localization of human beta- and deltaglobulin genes on chromosome 11, Proc. Natl. Acad. Sci. USA, 76 (1979) 4563-4565. 41 S6quier, J. and Lazdunski, M., Mast cell degranulating peptide induces the expression of the c-los proto-oncogene in hippocampus, Eur. J. Phramacol., 180 (1990) 179-181. 42 Sharp, F.R., Gonzalez, M.F., Hisangaga, K., Mobley, W.C. and Sagar, S.M., Induction of the c-los gene product in rat forebrain following cortical lesions and NGF injection, Neurosci. Lett., 100 (1989) 117-122. 43 Sharp, F.R., Sager, S.M., Hicks, K., Lowenstein, D. and Hisanaga, K., c-fos mRNA, Fos, and Fos-related antigens induction by hypertonic saline and stress, J. Neurosci., 11 (1991) 2321-2331. 44 Sheng, M. and Greenberg, M.E., The regulation and function of c-fos and other immediate early genes in the nervous system, Neuron, 4 (1990) 477-485. 45 Sickles, D.W., Toxic neurofilamentous axonopathies and fast anterograde axonal transport. I. The effects of single doses of acrylamide on the rate and capacity of transport, Neurotoxicology, 10 (1989) 91-101. 46 Sickles, D.W., Fowler, S.R. and Tetino, A.R., Effects of neurofilamentous axonopathy-producing neurotoxicants on in vitro production of ATP by brain mitochondria, Brain. Res., 528 (1990) 25-31.
236 47 Sonnenberg, J.L., Rauscher Ill, F.J., Morgan, J.l. and Curran, T., Regulation of proenkephalin by Fos and Jun, Science, 246 (1989) 1622-- 1625 48 Spencer, P.S. and Schaumburg, H.H., A review of acrylamide neurotoxicity. Part I. Properties, uses and human exposure, Can. J. Neurol. Sci., 1 (1974) 143-150. 49 Spencer. P.S. and Schaumburg, H.H., A review of acrylamide
neurotoxicity. Part 11. Experimental animal neurotoxicity and pathologic mechanism, Can. Z NeuroL Sci., 1 (1974) 152-169. 50 Spencer, P.S. and Schaumburg, H.H., Ultrastructural studies of the dying-back process. IV. Differential vulnerability of PNS and CNS fibers in experimantal central-peripheral distal axonopathies, J. Neuropathol. Exp. Neurol., 36 (1977)300-320.
Brain Research, 609 (1993) 231-236 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 18744
Acrylamide induces immediate-early gene expression in rat brain H. E n d o a, M.I. S a b r i b, J . M . S t e p h e n s c, P . H . P e k a l a c a n d S. K i t t u r a a Gerontology Research Center, Laboratory of Biological Chemistry, Baltimore, MD (USA), b Center for Research on Occupational and Environmental Toxicology, Oregon Health Sciences University, Portland, OR (USA) and c Department of Biochemistry, East Carolina University, Greenville, NC (USA) (Accepted 17 November 1992)
Key words: Acrylamide; c-los; c-jun; mRNA; Chemical injury; Neurotoxicity
Northern blot analysis was used to study the effects of acrylamide, a potent neurotoxin, on the induction of c-los and c-jun mRNA in rat brain. Male Sprague-Dawley rats (10-12 weeks old) treated with acrylamide as a single dose (100 mg/kg, i.p.) or via drinking water (0.03% w/v) for 4 weeks, were used to study acute and chronic effects on immediate-early gene expression, respectively. Acute administration of acrylamide caused a statistically significant increase in the expression of c-fos (approx. 37%) and c-jun (approx. 17%) mRNA in rat brain. By contrast, the level of c-los mRNA in chronic acrylamide treatment was not altered significantly, but the expression of c-jun mRNA was increased almost 100% as compared to control. These data show that the neurotoxin acrylamide induces immediate-early gene expression in the brain. The effects appear to be related to the route of administration, dose and duration of acrylamide treatment.
INTRODUCTION
c-fos mRNA in the ipsi- and contralateral hippocam-
The proto-oncogenes, c-fos and c-jun, and the respective proteins (Fos and Jun) are rapidly and transiently expressed in response to external stimuli in many ceils types, including neurons 32. Recent studies have shown that c-los mRNA and protein are induced in neurons and glia following brain injury 9'1°'42, hypoxia-ischemia and seizures 19 and activation of Nmethyl-D-aspartate receptor 1. c-fos has been proposed to function as a 'third messenger' molecule in the signal transduction system 32. Fos and Jun proteins are induced by neurotrophic factors, neurotransmitters, depolarizing conditions and agents that cause a voltage-gated calcium influx in PC12 cells 32. c-los is also induced in CNS neurons of intact animals by pharmacological, electrical, surgical, and physiological stimuli u. The level of c-los mRNA and corresponding protein induction is known to increase transiently in the cerebral cortex and the hippocampus after generalized seizures 31. Intrahippocampal injection of the mast cell-degranulating peptide, a bee venom component acting on the K ÷ channel, causes the expression of
pus without generating convulsions 4t. Preproenkephalin gene is known to be regulated by Fos and Jun proteins; levels of preproenkephalin mRNA were increased 1-2 h post-seizure, subsequent to the rise in Fos and Jun 47. Fos immunohistochemistry provides a cellular method to label polysynaptically activated neurons and thereby map functional pathways 38. Alterations in the structure and expression of proteins involved in signal transmission are known to be associated with experimental and human cancer 2°. The response of proto-oncogenes to xenobiotics that compromise neuronal integrity is uninvestigated. Acrylamide neurotoxicity is a suitable animal model to study the expression of these genes because responses to systemic exposure of this neurotoxin are characterized and experimentally reproducible 4,13,15,34,39,48-5°. Acute exposure leads to encephalopathy 22. Repeated intoxication with smaller doses of acylamide causes an ataxic syndrome in humans 14, neuronal loss 3'5 and distal axonal degeneration 34,48-5°. Chronically treated rodents lose microtubule-associated proteins (MAP1 and MAP2) and show dendritic changes in hippocampal
Correspondence: M.I. Sabri, Center for Research on Occupational and Environmental Toxicology (L606), Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR, USA. Fax: (1) (503) 494-4278.
232 and extrapyramidal areas ~'7. Chronic exposure also causes retrograde degeneration of long and large-diameter axons in gracile tracts and peripheral nerves, with associated sensory loss and motor weakness 39'49'50. While it is commonly suggested that axonal neuropathy results from a direct action of acrylamide on elements required for intracellular transport 2"3°'36'37'45"46,the possibility of a toxic effect on the neuronal perikaryon has been rarely investigated. This communication reports that acrylamide alters the expression of the immediate-early genes c-los and c-jun in rat brain. MATERIALS AND METHODS
Acute acrylamide treatment Sprague-Dawley rats (Charles River, Wilmington, MA) were treated with a single i.p. injection of either saline (control) or acrylamide (100 mg/kg). Rats were sacrificed by decapitation after 6 h, the brains excised, frozen on dry-ice and kept at - 80°C until used for RNA isolation. A time of 6 h was chosen for acute study since acrylamide causes optimum inhibition of fast retrograde axonal transport at this time 3°. Cerebral cortex from 5 acrylamide-treated and 3 saline-treated control rats were used for total RNA extraction.
Data were normalized to ~-actin mRNA. ¢¢-actin, an ubiquitous protein, was used as an internal control since total levels of this abundant protein were not unaltered by acrylamide. The following probes were used for these studies: (t) c-los; a l.(l-kb Pstl fragment of pfos-1 (ATCC, Rockville, MD)S; (2)c-jun; a 1.8-kb EcoRl/HindIII fragment (obtained from Dr. W.W. Lamph, The Salk Institute, San Diego)28; (3) /3-actin; 0.7-kb TaqI-Mcol cDNA fragment (Oncor, Gaithersberg, MD). RESULTS
Acute acrylamide treatment Autoradiograms of Northerns from control and acrylamide-treated samples hybridized with radiolabeled cDNA probes for c-fos, c-jun, and /3-actin are shown in (Fig. 1). Fig. 2 illustrates that the amount of c-fos mRNA (2.2 kb) was significantly increased (approx. 37%) following acrylamide treatment. The values of c-fos in acrylamide-treated (196.7 + 28.4) and control (143.2 + 9.0) samples were plotted as percent of /3-actin mRNA (Fig. 2). The values for/3-actin mRNA in acrylamide-treated (45.7 + 6.3) and control (45.8 + 0.5) samples show no significant difference in the expression of/3-actin message.
Chronic acrylamide treatment Rats were given 0.03% (w/v) acrylamide in drinking water for 4 weeks when all animals developed hind-limb weakness. Control and acrylamide-treated animals were decapitated, the brains excised, frozen on dry-ice and kept in -80°C until used for RNA extraction. Cerebral cortex from 4 acrylamide-treated and 3 saline-treated control rats were used for RNA extraction.
ACUTE ACRYLAMIDETREATMENT AC AC AC AC AC CT CT CT
RNA extraction and Northern blot analysis Brain tissues were homogenized with RNAzol B reagent (Cinna/Biotex, Friendswood, TX) (2 ml/100 mg). Homogenates were treated with 10% (v/v) chloroform, centrifuged at 12,000 × g for 15 min in a microfuge and the aqueous phase transferred to a fresh tube. An equal volume of 2-isopropanol was added, the mixture centrifuged at 12,000× g for 15 min whereupon a white-yellow pellet of RNA at the bottom of tube was obtained. The supernatant was removed, the RNA pellet washed with 75% ethanol by vortexing, and the material centrifuged for 8 rain at 7,500x g. Finally, the pellet was dissolved in diethyl pyrocarbonate-treated water. The concentration of RNA was determined spectrophotometrically by the absorbance at 260 nm. Ten micrograms of total RNA were denatured in formaldehyde and fractioned on 1% agarose gel. Size-fractionated RNA was transferred to a Nytran (Schleicher and Schuell, Keene, NH) membrane by capillary blotting and heated at 80°C. Filters were prehybridized at 46°C for 4 h in 50% (v/v) formamide containing 4 x SSC (150 mM sodium chloride, 15 mM sodium citrate), 1% SDS, 0.1% each of bovine serum albumin, polyvinylpyrrolidone, and Ficoll, 50 mM sodium phosphate, pH 7.4, and 100 tzg of yeast tRNA/ml of 0.5 mg/ml sodium pyrophosphate. Hybridization was carried out overnight in prehybridization solution containing 0.02% each of bovine serum albumin, polyvinylpyrrolidone and Ficoll, and either the c-los, c-jun, or /3-actin cDNA probe (1 × 107 cpm/ml) 4°. All cDNA probes were labeled with [32p] dATP (300 Ci/mmol) by random priming as described by Feinburg and Volgelstein12. After hybridization, the filters were washed in 0.1 x SSC, 0.1% SDS for 1 h at 65°C with constant agitation and then subjected to autoradiography using Kodak XAR-5 film in Dupont Cronex cassettes with intensifying screens for various periods at -80°C to ensure linear film response. The autoradiograms were developed and analyzed by a scanning laser densitometer. Differences in absorbance were quantified by Student's t-test. The same filters were stripped and reused for hybridization with other probes.
C-fos
c4un
9 i!ili if
~-actin
Fig. 1. Autoradiograms of RNA blot hybridized with 32p-labeled cDNA probes for c-los and c-jun and /3-actin from the cerebral cortex of acute acrylamide-treated (AC) and saline-treated control (CT) rats. Ten/zg of total RNA was loaded in each lane.
233 c-foe mRNA in acute acrylamide treatment
I
q
CHRONIC
ACRYLAMIDE
TREATMENT
AC AC AC AC CT CT CT
3oo 200 C-fos 100 ¸
=g
=_,..=
=erylamldetreated rats
controls
Fig. 2. Levels of c-los m R N A in acute acrylamide-treated and saline-treated control rats. Levels of the gene are expressed as a percent of /3-actin mRNA. Level of c-los mRNA in acrylamide treatment was significantly different from that in control ( P < 0.05). The amount of/3-actin m R N A in acrylamide-treated sample was not significantly different from controls.
Acute acrylamide treatment also significantly increased (approx. 17%) the induction of c-jun mRNA (2.7 kb and 3.2 kb) compared to control. The values of c-jun mRNA expressed as percent of/3-actin mRNA in acrylamide-treated and control samples were 74.6 + 10.9 and 63.7 + 0.1, respectively (Fig. 3).
c-jun
I~-actin
Chronic acrylamide treatment Fig. 4 shows autoradiograms of Northerns hybridized with radiolabeled c-fos, c-jun, and /3-actin cDNA probes. The level of c-fos mRNA in acrylamide-treated rats was not altered significantly compared to controls (Fig. 5). The values for c-fos mRNA in acrylamide-treated and control rats were 146.7 + 24.9 and 183.7 ___19.9, respectively (Fig. 5).
Fig. 4. Autoradiograms of RNA blot hybridized with 32P-labeled cDNA probes for c-fos and c-jun and ,8-actin from the cerebral cortex of chronic acrylamide-treated (AC) and saline-treated control (CT) rats. T e n / z g of total RNA was loaded in each lane.
By contrast, the level of c-jun mRNA in chronic acrylamide treatment (131.7 + 14.4) was significantly increased (100%) compared to control (65.9 + 11.7)
c-foe mRNA In chronic acrylamide treatment
c-jun mRNA in acute acrylamide treatment
!
3
lOO
|
80
,1, w
=
60 ,o,
o
r
, tuut=
acrylamlds, controls treated rate
Fig. 3. Levels of c-jun m R N A in acute acrylamide-treated and saline-treated control rats. Levels of the gene are expressed as a percent of fl-actin mRNA. Level of c-jun m R N A in acrylamide treatment was significantly increased compared to control ( P < 0.05). The amount of/3-actin m R N A in acrylamide-treated sample was not significantly different from controls.
-.in-
o
s©rylamldotreated rats
controls
Fig. 5. Levels of c-los m R N A in chronic acrylamide-treated and saline-treated control rats. Levels are expressed as a percent of /3-actin mRNA. Level of c-los m R N A in acrylamide-treated sample was not significantly different from that in control. The amount of fl-actin m R N A in acrylamide-treated sample was not significantly different from controls.
234
~<
200
c-jun mRNA in chronic ecrylamide treatment
U~Z
*. m Z~ Lu
-T" 100
iii ul .IL ecrylamldetreated rats
controls
Fig. 6. Levels of c-jun mRNA in chronic acr/lamide-treated and saline-treated control rats. Levels of c-jun are expressed as a percent of fl-actin mRNA. Level of c-jun mRNA in acrylamide-treated sample was significantly different from that in control (P < 0.05). The amount of fl-actin mRNA in acry]amide-treated samples was not significantly different from controls.
(Fig. 6). The values of/3-actin m R N A in chronic acrylamide-treated and control samples were 55.7 _+ 4.8 and 47.0 _+ 6.6, respectively, and are not significantly different from each other. DISCUSSION The results of this study show that systemic treatment with acrylamide induces immediate-early gene expression in rat brain. While the level of both c-los and c-jun m R N A was increased significantly following acute acrylamide treatment, only c-jun m R N A was elevated significantly following chronic acrylamide treatment. Increased mRNA could be the result of increased immediate-early gene transcription through the activation of a signal transduction system. Biochemical changes such as increased concentrations of NAD + in whole brain 25, elevated levels of 5-hydroxyindoleacetic acid in the striatum, septal area, and thalamus 35 and/3-glucuronidase activity have been reported in acrylamide-treated animals 5. Differential effects of acrylamide, given as a single injection or repeated doses, have been observed previously ~6'17. While a single injection of acrylamide produces a modest retardation of the slow axonal transport of neurofilaments, chronic acrylamide treatment causes blockade of neurofilaments, reduced axonal caliber in proximal axons, and distal axonal degeneration ~6'17. The induction of c-jun and not of c-fos after chronic treatment with acrylamide is very interesting in the light of previous studies showing selective induction of c-jun mRNA and Jun proteins in dorsal root ganglion neurons after sciatic nerve transection, crush or blockade of axonal transport with colchicine or
vinblastine 24'2~'43. These reports suggest that c-jun may play an important role in neuronal response to chemical and traumatic injury. c-los activation in neuronal cells occurs within minutes of growth factor stimulation and precedes the activation of c-myc 1~'27"33. The induction of c-fos is transient and disappears 30 min after stimulation 44. It is therefore not surprising that c-los induction is not seen following chronic acrylamide treatment. Reasons for a differential effect of acute and chronic acrylamide on the induction of immediate-early genes in rat brain is not immediately clear. However, these results should be compared with findings in ischemic animals where c-los expression was dramatically elevated 72 h post ischemic injury in the CA1 region. It was speculated that c-fos participates in the long-term alteration of cellular function in individual regions of the hippocampus following neuronal injury, and may also be involved in regeneration of nerve cell processes z6. It has been suggested that c-los plays a role in the maintenance of long-term changes by increasing synaptic activity 23. Since c-los and c-jun are thought to control physiological processes of cell growth and differentiation 21, their m R N A might be activated by acrylamide-induced pathophysiological changes. It appears therefore that acrylamide may affect neuronal signal transduction and that activation of immediate-early genes may be related to a compensatory mechanism to overcome acrylamide toxicity. In conclusion, the results of this study indicate that the expression of immediate-early genes (c-fos and c-jun) is up-regulated in the CNS following acrylamide treatment. While acute acrylamide administration induces both c-los and c-jun, chronic acrylamide treatment induces only c-jun. The results of this study suggest that IEGs may play important roles in neuronal responses to chemical injury.
Acknowledgements. We would like to acknowledge Mrs. Charlotte Adler for support with photography. Supported in part by NIH Grant NS 19611.
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